Disodium L-tyrosinate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 December 2017 - 25 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 (REACH). B.40.bis. In vitro skin corrosion: human skin model test.

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic™ Skin Corrosivity Test

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium L-3-(4-hydroxyphenyl)alaninate hydrate

Molecular formula:

C9H9NNa2O3 • xH2O

IUPAC Name:

Disodium L-3-(4-hydroxyphenyl)alaninate hydrate

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 16-RHE-130

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min or 1 h)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: gently rinsing with a minimum volume of 20 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer.
In the pre-test medium coloration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for color control were treated according to the INVITTOX Protocol SkinEthic™ Skin Corrosivity Test (2012).

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied : 20 ± 3 mg

NEGATIVE CONTROL
- Amount applied: 40 ± 3 µL

POSITIVE CONTROL
- Amount applied: 40 ± 3 µL

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

82.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

79.89

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Acceptability of the Test

Acceptability of the Negative Control: The negative control OD values were 2.019, 2.059, 1.675 and 1.699 and, thus, in the range of ≥ 0.8 and ≤ 3.0.

Acceptability of the Positive Control: After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.59 % after 1 hour exposure and, thus, lower than 15 %.

Test Substance Data Acceptance Criteria: The range between identically treated tissues was less than 30 % (27.10 % after 3 minutes exposure and 13.63 % after 1 hour exposure).

The study met all acceptance criteria

Table 1: Results

Group		Tissue 1		Tissue 2		Mean		CV
		OD	Viability (%)	OD	Viability (%)	OD	Viability (%)	Viability (%)
Negative Control	3 min	20.19	99.02	2.059	100.98	2.039	100.00	1.39
	1 h	1.675	99.30	1.699	100.70	1.687	100.00	0.99
Positive Control	1 h	0.009	0.51	0.011	0.66	0.010	0.59	17.90
Test Substance	3 min	1.481	72.66	1.883	92.35	1.682	82.50	16.88
	1 h	1.262	74.79	1.434	84.99	1.348	79.89	9.02

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the test item showed no skin corrosive potential.

Executive summary:

In an in vitro skin corrosion assay (RhE) according to OECD Guideline 431, the potential of the test item to induce skin corrosion in an in vitro human skin model was investigated.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

After treatment with the negative control (deionised water) the mean OD per tissue replicate was 2.019 and 2.059 after 3 minutes exposure and 1.675 and 1.699 after 1 hour exposure (study acceptance criterion: ≥ 0.8 and ≤ 3.0). Treatment with the positive control (Potassium hydroxide, 8N) revealed a mean viability of 0.59% after 1 hour (study acceptance criterion: < 15%). Therefore, the study fulfilled the validity criteria.

Following treatment with the test item, the tissue viability was > 50% after 3 minutes exposure (mean viability: 82.50%) and > 15% after 1 hour exposure (mean viability: 79.89%), i.e. according to OECD 431 the test item is considered as non-corrosive to skin.